Therapeutic opportunities in biological responses of ultrasound

*

and bones, the use of ultrasound for inducing non-necrotic tumor atrophy as well as for potentiation of chemotherapeutic drugs, acti-vation of the immune system, angiogenesis and suppression of phagocytosis. A review of these therapeutic opportunities is presented

2.2. Cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

Interactions of ultrasound with biological tissues have ledto several clinical therapies including physiotherapy [1], trans-

ment [4]. Most of these therapies are based on physicaleects of ultrasound on cells and tissues such as controlleddisruption of various biological barriers including cell mem-branes and tissues for drug and gene delivery [5,6]. In con-trast, relatively less is known about the therapeutic potentialof subtle, biological eects of ultrasound on cells and tissues.

* Corresponding author. Tel.: +1 805 893 7532; fax: +1 805 893 4731.E-mail address: samir@engineering.ucsb.edu (S. Mitragotri).

Available online at www.sciencedirect.com

Ultrasonics 48 (2008)2.3. Stimulation of immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2742.4. Inducing arteriogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2742.5. Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

3. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

1. Introduction dermal drug delivery [2], thrombolysis [3] and cancer treat-with particular emphasis on their mechanisms. Overall, this review presents the increasing importance of ultrasounds role as a biologicalsensitizer enabling novel therapeutic strategies.Published by Elsevier B.V.

Keywords: Ultrasound; Biological response; Wound healing; Bone regeneration; Cancer therapy; Immune response; Angiogenesis; Hemostasis

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2712. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

2.1. Tissue healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2722.1.1. Soft tissue stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2722.1.2. Bone regeneration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272The therapeutic benets of several existing ultrasound-based therapies such as facilitated drug delivery, tumor ablation and throm-bolysis derive largely from physical or mechanical eects. In contrast, ultrasound can also trigger various time-dependent biochemicalresponses in the exposed biological milieu. Several biological responses to ultrasound exposure have been previously described in theliterature but only a handful of these provide therapeutic opportunities. These include the use of ultrasound for healing of soft tissues0041-6

doi:10.ctAbstraReceived 1 July 2007; received in revised form 5 February 2008; accepted 28 February 2008Available online 7 March 2008Sumit Paliwal, Samir Mitragotri

Department of Chemical Engineering, University of California, Santa Barbara, CA 93106, United States24X/$ - see front matter Published by Elsevier B.V.

1016/j.ultras.2008.02.002www.elsevier.com/locate/ultras

271278

ltraMost organisms exhibit innate ability to respond to var-ious environmental insults so as to facilitate robust recov-ery and minimal long-term eects of the insult. In thiscontext, ultrasound also exerts non-lethal trauma to thebiological milieu and can incite a survival response. Biolog-ical responses of ultrasound on cells and tissues have beenpreviously addressed in the literature [7,8]. However, themajor focus in these studies had been to understand anddevelop safety standards for countering the possible delete-rious bioeects of ultrasound exposure [9]. For example,several reports describe ultrasounds biological eects onembryos, central-nervous system, and other major organsat a cellular and molecular level [8,10]. Relatively little isknown about the benecial biological responses ofultrasound. Over the last years, a few therapies based onultrasounds biological eects have been demonstratedwith specic applications in tissue regeneration, cancertreatment, and immune and vasculature stimulation. Thisarticle reviews these novel approaches especially the under-lying connections between ultrasound-induced biologicalresponses and therapeutic ecacy.

2. Applications

2.1. Tissue healing

With a history spanning over 6 decades, stimulation ofinjured tissues for accelerated repairing is the most studiedand clinically practiced biological response of ultrasound[9]. Therapeutic eects of regular ultrasound exposure havebeen described in the literature for repairing damagedligaments, muscle spasms, inamed tendons, sti joints,fractured bones and cartilage [1113]. Related eects havealso been used for debridement and accelerated healing ofwounds [1417], skin rejuvenation [18,19], nerve stimula-tion [2022], and improving the strength and elasticity ofscar tissues [23]. Below, we discuss some of the prominentapplications of this therapy and their underlyingmechanisms.

2.1.1. Soft tissue stimulation

Ultrasound, through its thermal and non-thermal (cavi-tation and acoustic streaming) mechanisms, has been impli-cated in stimulating soft tissue healing. Tissues, particularlythose containing densely-packed large protein moleculessuch as collagen, can experience elevated temperaturesresulting in several therapeutic benets. These includeincreased extensibility/exibility of collagen-rich scar tis-sues [23], tendons [24,25] and joints [26], pain and spasmrelief due to heating of muscles [27] and nerve roots [2022], and possible increase in blood ow [28] to help resolu-tion of chronic inammatory processes. Role of non-ther-mal mechanisms of ultrasound in tissue regeneration [29]and soft tissue repair [17,3032] has also been widely estab-lished. At a cellular-level, it has been hypothesized that

272 S. Paliwal, S. Mitragotri / Uchanges in diusion rates and membrane permeability toions [3335] due to acoustic streaming and stable cavita-tion, can stimulate cells by upregulation of signaling mole-cules [11]. Specically, ultrasound has been shown toincrease protein synthesis [31] including that of collagen[32,36], which is essential for repair mechanisms in cells.During the inammation phase of the healing process,ultrasound can activate immune cells to migrate to the siteof injury. In two separate studies, Fyfe et al. showed ultra-sonic induction (0.5 W/cm2) of mast cell degranulation andhistamine release in injury models in vivo [37,38]. Similarresults were reported for dermal mast cells, demonstratingthat ultrasound can accelerate the inammatory healingphase for skin lesions/ulcers in vivo (Wistar rats, 0.75-3 MHz, 0.25-3 W/cm2) [39]. In a related study, Younget al. showed that ultrasound (3 MHz, 0.5 W/cm2) canstimulate macrophages in vitro to release broblast mito-genic factors, resulting in enhanced broblast proliferation[40]. Ultrasound may also help in wound contraction [41]and scar tissue remodeling [42] by possibly altering the col-lagen ber pattern. These benecial eects of ultrasoundhave been used in treating various skin conditions includ-ing varicose ulcers [17], skin lesions [43], pressure sores[30,44], acceptance of skin grafts [45], and sutured incisedwounds [46]. Therapeutic eects due to ultrasound expo-sure have been demonstrated during regeneration of dam-aged muscle tissue [27], and peripheral and sciatic nerves[2022]. In an exciting application, ultrasound has beenproposed for skin rejuvenation and wrinkle removal bycontrolled ablation of dermal tissue and provoking awound healing type response [18,19].

2.1.2. Bone regeneration

In addition to the various tissue repair applications andmechanisms discussed above, ultrasonic bone regenerationforms an attractive subset due to bone tissues special phys-iology and the clinical success of this therapy. Stimulatoryeect of ultrasound on osteogenesis was established asearly as in 1950 [47], but its clinical popularity as a thera-peutic tool for treating fractures came much later in1990s (1994: FDA approval for ultrasonic treatment offresh fractures). This was facilitated by the use of low-intensity pulsed ultrasound (LIPUS; early report Ref.[48]). Many studies have demonstrated the use of ultra-sound for accelerating healing in fresh [49,50], nonunion[5153] and delayed-union [53] type fractures, and forenhanced ossication in distraction osteogenesis [5456].Healing of fractures is a complex process involving inam-mation, proliferation (angiogenesis and callus formationand hardening), and remodeling of the bone tissue. Plentyof evidence supports the conclusion that ultrasound ismore eective in early-stage of fractures (inammationand proliferation) compared to the remodeling phase [5761]. While during the inammation phase, formation ofhaematoma helps recruitment of cells to the fracture site,the proliferation phase includes angiogenesis, dierentia-tion of mesenchymal stem cells and endochondral ossica-

sonics 48 (2008) 271278tion [62]. In vitro ultrasonic treatment of cells that aretypically involved in fracture healing (osteoblasts, bro-

ltrablasts and monocytes), showed secretion of inammatorycytokines (IL-1b) and angiogenesis-inducing factors (IL-8, b-FGF, and VEGF) inducing cell proliferation, collagenproduction, bone formation, and angiogenesis [6365].Rawool et al. demonstrated a 33% increase in vascularityat the osteotomy site in dogs, suggesting that ultrasound(1.5 MHz, 30 mW/cm2: daily treatment for 11 day) maystimulate angiogenesis, which is critical for successfulosteogenesis [66]. Several studies have also indicated thatultrasound signicantly stimulates the formation andstrengthening of calluses, which was demonstrated by fastrecovery of the mechanical properties of bone tissue (mea-sured by bones maximal torque and torsional stiness)[48,57,59,61]. Tsai et al., using fractured rabbit bulaemodel, showed enhanced mineral apposition rates in ultra-sound-treated cases (1.5 MHz, 0.5 W/cm2: treatment for525 min/day) [67]. Further, dierentiation of cartilage-specic chondrocytes from mesenchymal stem cells [6870]and subsequent upregulation of various biomolecules suchas procollagens [71], aggrecan [59,70,71], proteoglycan [71],and chondroitin sulphate [72] as a response to ultrasoundhave been reported. Interestingly, ultrasound has also beendemonstrated in vitro to directly stimulate bone formation,in terms of quantitative increase in various bone markersincluding osteocalcin and alkaline phosphatase [7375].

2.2. Cancer therapy

Extensive work has been done in the past showing harm-ful biological responses of cells and tissues to ultrasoundexposure including growth suppression, retarded proteinsynthesis, cytoplasmic vacuolation and disruption of intra-cellular components such as mitochondria, microtubulesand endoplasmic reticulum (for a comprehensive compila-tion of such eects, see Ref. [10]). In clinical practice suchharmful and potentially lethal biological eects of ultra-sound can be used to target tumor suppression or possiblyelimination. Specically, ultrasound-induced microtubuledisassembly [7678] is of special interest in cancer therapy.Cancer drugs such as taxol, which are known to disruptintracellular microtubule dynamics, are routinely used forchemotherapy. Hrazdira et al. reported that a low-intensityultrasound exposure (0.8 MHz, 50500 mW/cm2 for510 min) made HeLa cells undergoing M- and S-phasesof the cell cycle sensitive to cytoskeleton disassembly withprominent changes in the microtubules and microlamentsnetworks [78]. Later, Skorpikova et al. using similar ultra-sound parameter, followed up with these results to showthat ultrasonic cytoskeletal disruption in tumor cells poten-tiated two cytostatic cancer drugs, cycloplatin and metho-trexate [77].

Another application of ultrasound is to induce non-necrotic and localized hyperthermia in tumor tissues forcancer therapy [79,80]. Sustained heating of solid tumor tis-sues at temperatures of 42.543 C for 2030 min is known

S. Paliwal, S. Mitragotri / Uto induce favorable therapeutic eect [81]. Several clinicalstudies have successfully demonstrated tumoricidal eectsof ultrasonic hyperthermia employing frequencies in therange of 13 MHz [8284]. It is understood that ultrasonichyperthermia may manifest its therapeutic eects througharresting cancer cells in the S phase of the cell cycle andmay have preferential eects on hypoxic, acidic and mal-nourished tumor cells [79].

Several clinical studies have been actively undertakenfor the treatment of solid tumors in prostate [85,86], liver[87], breast [88], kidney [89], sarcoma [87] and uterus [90]using ultrasound-induced hyperthermia. While instanta-neous thermal ablation remains the chief mechanism of thistreatment strategy, recent in vivo studies have indicatedthat ultrasound exposure can strongly stimulate long-termantitumor immunity in the host. In a preliminary study,Yang et al. demonstrated that ultrasound exposure(4 MHz, 550 W/cm2) of neuroblastoma implanted in miceled to signicantly slower growth of the tumor when itwas re-implanted at the previously ultrasound-treated site[91]. The authors concluded that ultrasound exposure acti-vated the immune response against the tumor. Mechanisti-cally, ultrasound treatment can facilitate the interactions oftumor-specic antigens with the immune system and elicitan antitumor response. Wu et al., in two separate studieson 23 breast cancer patients, showed that several tumorantigens, which are otherwise sequestered in the tumor-cytoplasm, were extensively present, preserved and avail-able for immunostimulation in the extracellular matrix ofthe ultrasound-ablated (1.6 MHz, 515 kW/cm2) tumordebris [92,93]. Further, ultrasound exposure can increasethe tumor antigenicity by release of endogenous inamma-tory cytokines [94,95] as well as upregulation of heat shockproteins [93,95,96] in the surrounding viable tissues, andthereby activating and attracting immune cells to the tumorsite. Hu et al. showed that ultrasound exposure at 1.1.MHz to MC-38 colon adenocarcinoma tumor cellsin vitro led to the release of endogenous ATP and hsp60in the culture medium [94]. Furthermore, exposure of thisculture medium resulted in robust activation of antigen-presenting cells (dendritic cells and macrophages) and ledto enhanced secretion of pro-inammatory cytokines(IL-12 and TNF-a) by them [94]. In a follow up study,Hu et al. ablated MC-38 tumors in mice and found pro-nounced accumulation of activated dendritic cells in theultrasound-treated tumors and in the local draining lymphnodes [97]. Such inammatory reaction upon ultrasoundexposure can provide an ideal environment for the develop-ment of mature T-cells and lead to a systemic antitumorimmune response. Rosberger et al. reported for patientswith posterior choroidal melanomas that ultrasoundtreatment resulted in signicant increase in CD4 T cells[98]. In a total of 16 patients with osteosarcoma, hepatocel-lular carcinoma, or renal cell carcinoma, Wu et al. reportedthat ultrasound-assisted tumor ablation (0.8 MHz, 520 kW/cm2) resulted in a signicant increase in the popula-tion of CD4+ lymphocytes and cytotoxic NK cells in the

sonics 48 (2008) 271278 273peripheral blood [99]. Interestingly, a recent study by Zhouet al. demonstrated that ultrasound treatment can augment

ltraantitumor immunity by reducing the immunosuppressiveeects of tumors a phenomenon by which cancer cellscan downregulate hosts immune system, resulting in theirenhanced growth and progression [100]. The authorsreported that serum levels of immunosuppressive cytokines(VEGF, TGF-b1, and TGF-b2) in patients with solidmalignancies were signicantly reduced after ultrasoundtreatment (0.8 MHz, 520 kW/cm2) [100].

Inducing selective cytotoxicity in tumor cells, while leav-ing healthy cells unaected, is a signicant challenge.Through in vitro studies, using biological responsestowards ultrasound, Paliwal et al. showed that a brief expo-sure to ultrasound (20 kHz, 2 W/cm2) followed by treat-ment with quercetin (a weak chemotherapeutic drug)induced selective cytotoxicity in prostate and skin cancercells [101]. The investigators reported that about 90% ofthe skin cancer cell population was lost after ultrasoundapplication and subsequent 48 h incubation with 50 lMquercetin. Further, ultrasound led to an 80-fold enhance-ment in skin cancer cell toxicity (measured by LC50 ofquercetin), while the treatment had minimal eects onnon-malignant skin cells. This high treatment selectivitytowards cancer cells was hypothesized to be due to thedierential protein-expression response of heat shockprotein-hsp72 after ultrasound exposure. hsp72 is a vitalstress-protein essential for the survival of cancer cells; theultrasound-quercetin combined treatment induced selectiveand strong inhibition of hsp72 in cancer cell as compared tonormal cells [101].

2.3. Stimulation of immune response

Transcutaneous immunization (TCI) involves applica-tion of vaccines on the skin to induce immunization[102]. Physiologically, skin represents bodys rst line ofdefense against pathogens and hence it is an ideal site forvaccination due to the presence of various immune cells,particularly Langerhans cells (LCs) which are highly potentantigen-presenting cells [103]. However, simple topicalapplication of vaccines does not yield an adequate immuneresponse [102]. In order to achieve a robust immuneresponse through TCI, skin tissue has to be sucientlystimulated with adjuvants to develop an inammatory typeresponse, which includes secretion of signaling proteins cytokines leading to the activation of immune cells[104,105]. In parallel to pro-inammatory toxic chemicaladjuvants, ultrasound has been demonstrated as a potentphysical adjuvant for TCI. Specically, pre-exposure ofultrasound (20 kHz, 2.4 W/cm2) followed by topical incu-bation of tetanus toxoid resulted in strong systemicincrease of specic IgG antibody titers in mice, which werecomparable to the titers obtained from conventionallyadministered subcutaneous vaccine injections, though atmuch higher doses [106]. The mechanisms behind this novelbiological response of ultrasound to stimulate skins

274 S. Paliwal, S. Mitragotri / Uimmune functionality have been studied. Ultrasound,through acoustic cavitation, breaches the skins barrier[107], which can result in skins inammatory responseneeded for TCI. Indeed, the connection between restora-tion of skin permeability barrier homeostasis and activationof various signaling pathways leading to upregulation ofvarious pro- inammatory cytokines and growth factors,is well established [108]. This hypothesis was revealed ina study by Choi et al., who showed that 1 MHz ultrasoundexposure up-regulates epidermal pro-inammatory cyto-kine (IL-1a, TNFa, and TGFb) expression in skin [109].Ultrasound exposure activated various immune cells inthe skin, particularly Langehans cells [106]. Overall, theseresults suggest that ultrasound, in its capacity as a mechan-ical stimulant, can enable upregulation of cytokines in theskin, activate immune cells and generate an immuneresponse against topically delivered vaccines.

Intravenously-injected polymer nanoparticles havefound applications in several medical therapies, particu-larly due to the high exibility in tuning their propertiessuch as size, shape, surface characteristics and degradationrate [110]. Nanoparticles are being developed as contrastagents for blood and tissue imaging, with specic applica-tion in cancer diagnosis [111]. Amid considerable interest,polymer nanoparticles have also been used for drug deliv-ery applications [112]. In their role as carriers of therapeu-tic agents, nanoparticles provide several distinctadvantages such as enzymatic protection of the drug,targeted delivery and sustained release of the drug overprolonged time periods [113115]. In spite of this greatpotential, nanoparticles have found limited practical appli-cations due to their short vascular circulation lifetimes[116,117]. Circulating particles, however, are recognizedand captured by the phagocytic cells of the immune system(reticuloendothelial system) within minutes of their admin-istrating [110,116]. Ultrasound has been previouslyreported to suppress the phagocytic activity of immunecells, and thus can nd a potentially attractive therapeuticapplication as an extracorporeally-administered and tar-geted immuno-suppressant for prolonged circulation ofpolymeric particles. In a direct attempt to evaluate ultra-sonic response of the reticuloendothelial system, Saadet al. demonstrated in vivo that ultrasound exposure tothe umbilical area of rats showed decreased clearance ofintravenously-injected colloidal sulfur particles [118]. Theclearance was shown to further decrease with increasingintensity of ultrasound and with the duration of exposure(1.65 MHz, 60400 J/cm2) [119]. Studies by Andersonet al. also showed impaired anti-bacterial activity of perito-neal macrophages [120], depression of phagocytic clearanceof intravascular carbon [120], and immuno-suppressiveeects on spleen [121] by ultrasound exposure (2 MHz,8.9 mW/cm2).

2.4. Inducing arteriogenesis

Occlusive vascular disease is marked by inadequate

sonics 48 (2008) 271278blood ow to organs often leading to malnutrition andischemic conditions, particularly involving the myocar-

ltradium and skeletal muscle tissue. Various approaches fortargeted stimulation of neovascularization have beenpursued in the past, including delivery of growth factorproteins or genes [122] and more relevantly, by inducinginammatory response in the aected vasculature [123].In this context, ultrasound was hypothesized to initiatean inammatory response through implosion of microbub-bles in ischemic tissues, leading to triggering of variousangiogenic pathways [124]. In this study, exposure of1 MHz ultrasound to intravenously-injected microbubblesresulted in increased arteriole density, arteriole diameters,and nutrient blood ow into the treated rat skeletal muscle[124]. With a similar procedure, microvascular remodelingresponse was observed in a rat model of arterial occlusion[125]. In a recent mechanistic study, the therapeuticpotential of this strategy was shown to originate from therecruitment of growth factor-producing cells to the ultra-sonically-inamed site [126]. Interestingly, a combinedtreatment of ultrasound-triggered arteriogenesis and trans-plantation of bone marrow-derived mononuclear cellsfacilitated blood ow restoration by stimulating bothangiogenesis and arteriogenesis in an ischemic rat model[127,128].

2.5. Hemostasis

High-intensity focused ultrasound (HIFU), at typicalexposure settings of 15 MHz and 110 kW/cm2, has beensuccessfully demonstrated to achieve hemostasis in activebleeding from blood vessels [129131] and organs such asliver [132,133] spleen [134] and lung [135]. In a pig model,using 2 MHz and 3.5 MHz HIFU transducers operatingat 0.53.1 kW/cm2, Vaezy et al. reported cessation ofbleeding from several types of punctured blood vesselsincluding abdominal aorta, femoral artery and vein, axil-lary artery, carotid artery and the jugular vein [130]. Trea-ted vessels showed localized hardening of the soft tissuesurrounding the vasculature and coagulation throughextensive brin network around the vessels, providing aseal for the punctured hole [130]. Zderic et al. examinedthe long-term eects of HIFU-assisted hemostasis in punc-tured femoral arteries of rabbits [131]. Prolonged hemosta-sis over a period of 60 days was observed with no dierencein blood ow velocities and hematocrit levels at the end ofthe study [131]. Long-term safety of ultrasound to providehemostasis in acute organ injuries to the spleen and liverhas been also established in animal models [132,134].

Ultrasonic hemostasis strategy employs a high-intensitybeam of ultrasound, which is focused on the ruptured vas-culature to achieve rapid heating (tissue temperatureexceeding 70 C), leading to coagulative necrosis of thetissue and hemostasis [136,137]. Although this apparentcauterization of the tissue should be classied as a directthermal bioeect of ultrasound, several post-exposureobservations reveal active recovery of the injured site a

S. Paliwal, S. Mitragotri / Ubiological response of ultrasound exposure resulting inhealing and prolonged hemostasis. For example, Vaezyet al. reported on post-operative wound healing type recov-ery response after ultrasonic hemostasis of surgical liverinjury in rabbit model [133]. They observed dense collagendeposition and signicant tissue regeneration (indicated bygrowth of new hepatocytes) at the treated site within 60days of therapy. It was hypothesized that as early as 2weeks after ultrasound exposure, the treatment can activateleukocytes and broblasts from neighboring areas tomigrate and produce collagen, elastin and proteoglycansto enhance healing [133]. Additional studies have indicatedthat hemostasis may also be boosted by tissue thrombo-plastins released from ultrasonically heated endothelium[138]. It is understood that tissue recovery responses inhemostasis can be mediated by the non-thermal mecha-nisms of ultrasound, particularly acoustic cavitation. Poli-achik et al. used ultrasound contrast agents to demonstratethat cavitation can induce platelet activation, aggregationand adhesion to collagen coated surfaces a phenomenonnecessary for platelet plug formation in hemostasis [139].Further, ultrasound exposure was shown to disrupt plate-lets to release b-thromboglobulin, adenosine diphosphateand other chemical factors at the site of bleeding, whichcan induce clotting as well as recruitment of undamagedplatelets for accelerated clot formation [140,141].

3. Conclusions

Ultrasound nds much of its therapeutic applicationsfrom thermal, physical and mechanical eects on cellsand tissues. Such direct eects of ultrasound can inducestresses in cells and tissues leading to several long-termrecovery responses. Increasingly, such ultrasound-inducedbiological responses are being innovatively engineered totherapies including tissue healing and rejuvenation, cancertreatment, immune adjuvancy, arteriogenesis and hemosta-sis. At a tissue level, most of the observed ultrasonic ther-apeutic benets are mediated by an inammatory responseresulting in a coordinated recruitment of cells as seen inaccelerated tissue regeneration, activation of immune cellsin vaccination, or in stimulated growth of vasculature. Sev-eral cellular responses to ultrasound have been reported,but only a handful of these eects such as upregulationof key proteins have been therapeutically implicated.Mechanistically, the exact biochemical pathways behindultrasonic therapies are not completely clear and oftenmany studies in the literature report conicting results.For example, ultrasound has been reported to activate epi-dermal immune cells, while direct ultrasound exposure toimmune cells has been also demonstrated to suppress theiractivity. Inconsistencies in the literature might arise due tousage of dierent ultrasound parameters resulting in acti-vation of dierent biochemical pathways (such as apoptosisvs. necrosis). Another source of discrepancies is thediculty in transforming ex vivo (cellular-level) results toanimal-level studies due to signicant dierences in ultra-

sonics 48 (2008) 271278 275sonic bioeects at a tissue level and actuation of homeo-static mechanisms in vivo. Although, ultrasonic therapy

peripheral nerve regeneration in poly (DL-lactic acid-co-glycolic

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sonics 48 (2008) 271278

Therapeutic opportunities in biological responses of ultrasoundIntroductionApplicationsTissue healingSoft tissue stimulationBone regeneration

Cancer therapyStimulation of immune responseInducing arteriogenesisHemostasis

ConclusionsReferences